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Clostridium perfringens causes a variety of human and animal enteric diseases including food poisoning, antibiotic-associated diarrhea, and necrotic enteritis. Yet, the reservoirs of enteropathogenic enterotoxin-producing strains remain unknown. We conducted a genomic comparison of 290 strains and a heat resistance phenotyping of 30 C. perfringens strains to elucidate the population structure and ecology of this pathogen. C. perfringens genomes shared a conserved genetic backbone with more than half of the genes of an average genome conserved in >95% of strains. The cpe-carrying isolates were found to share genetic context: the cpe-carrying plasmids had different distribution patterns within the genetic lineages and the estimated pan genome of cpe-carrying isolates had a larger core genome and a smaller accessory genome compared to that of 290 strains. We characterize cpe-negative strains related to chromosomal cpe-carrying strains elucidating the origin of these strains and disclose two distinct groups of chromosomal cpe-carrying strains with different virulence characteristics, spore heat resistance properties, and, presumably, ecological niche. Finally, an antibiotic-associated diarrhea isolate carrying two copies of the enterotoxin cpe gene and the associated genetic lineage with the potential for the emergence of similar strains are outlined. With C. perfringens as an example, implications of input genome quality for pan genome analysis are discussed. Our study furthers the understanding of genome epidemiology and population structure of enteropathogenic C. perfringens and brings new insight into this important pathogen and its reservoirs.

Correction: Volume: 6 Issue: 5 Article Number: e00733-21 DOI: 10.1128/mSphere.00733-21 Listeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry. IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen's transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Clostridium botulinum group III is the anaerobic Gram-positive bacteria producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol to obtain high-molecular-weight DNA from C. botulinum group III is described.

Clostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages&mdash, one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Listeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry. IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen’s transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Listeria monocytogenes is a food-borne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements of Listeria inhabiting dairy farms. We performed a SNP-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes that persistent on the farms for ≥ 6 months. We found that prophages and other mobile genetic elements were on average more numerous among isolates in persistent than nonpersistent clades, and demonstrated that resistance genes against bacitracin, arsenic and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons and a novel prophage harbouring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of Enterococci, indicative of recent transfer between these genera. Finally, we demonstrated that the CRISPR-cas IIa system and a type II restriction-modification system were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry.ImportanceAnimal derived raw materials are an important source of L. monocytogenes for the food industry. Knowledge of the factors contributing to the pathogen’s transmission and persistence on farms are essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favourable phenotypes, such as resilience against biocides and thermal stress. Understanding the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to rise the novel resistance phenotypes.

To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HIC deletion mutant strains under heat (42.5 degrees C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H2O2) stresses. The growth of Delta liaS (Delta lmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The Delta ivirS (Delta lmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of Delta agrC (Delta lmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HIC mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L monocytogenes EGD-e. (C) 2017 Elsevier Ltd. All rights reserved.

Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni are notable health hazards associated with the consumption of raw milk. These bacteria may colonize the intestines of asymptomatic cattle and enter bulk tank milk via fecal contamination during milking. We studied the frequency of STEC O157:H7 and C. jejuni contamination in tank milk (n = 785) and the in-line milk filters of milking machines (n = 631) versus the frequency of isolation from cattle feces (n = 257) on three Finnish dairy farms for 1 year. Despite simultaneous isolation of STEC O157:H7 (17%) or C. jejuni (53%) from cattle, these bacteria were rarely isolated from milk filters (2% or

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31156582/ Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm's outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm's outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk. This research was supported by the Finnish Ministry of Agriculture and Forestry grant Dnro: 1395/311/214, the Walter Ehrström Foundation, and the University of Helsinki Doctoral Program in Food Chain and Health. The sequencing of the strains and the salary of AJ and JI were supported by the INNUENDO project (https://sites.google.com/site/theinnuendoproject/) which received funding from the European Food Safety Authority, grant agreement GP/EFSA/AFSCO/2015/01/CT2 (“New approaches in identifying and characterizing microbial and chemical hazards”). info:eu-repo/semantics/publishedVersion

Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control.

The activity of RNA helicase csdA ( cbo2802 ) after temperature downshift was compared to its activity at optimal growth temperature, and the effect of sense and antisense oriented insertional inactivation of cbo2802 on the growth of ATCC 3502 at suboptimal temperature was evaluated. The relative cbo2802 transcript level was significantly induced for 30 min to 5 h after cold shock. In contrast, a significant decrease in the relative transcript level of cbo2802 was observed within the same time frame at 37 °C. Inactivation of cbo2802 led to an extensive delay in initiation of exponential growth at 20 °C but not at 37 °C. In addition, the mean minimum growth temperatures of the mutant strains were higher than those of the wild-type strain. During a 24-hour incubation at 37 °C, all strains were motile, whereas at 20 °C the mutant strains showed severely impaired motility compared to the wild-type strain. This study shows that a functional csdA is needed for effective adaptation and initiation of growth and motility of Clostridium botulinum ATCC 3502 at suboptimal temperature.

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK , proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the Δ resE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.

Vegetative cultures of Clostridium botulinum produce the extremely potent botulinum neurotoxin, and may jeopardize the safety of foods unless sufficient measures to prevent growth are applied. Minimal food processing relies on combinations of mild treatments, primarily to avoid deterioration of the sensory qualities of the food. Tolerance of C. botulinum to minimal food processing is well characterized. However, data on effects of successive treatments on robustness towards further processing is lacking. Developments in genetic manipulation tools and the availability of annotated genomes have allowed identification of genetic mechanisms involved in stress tolerance of C. botulinum. Most studies focused on low temperature, and the importance of various regulatory mechanisms in cold tolerance of C. botulinum has been demonstrated. Furthermore, novel roles in cold tolerance were shown for metabolic pathways under the control of these regulators. A role for secondary oxidative stress in tolerance to extreme temperatures has been proposed. Additionally, genetic mechanisms related to tolerance to heat, low pH, and high salinity have been characterized. Data on genetic stress-related mechanisms of psychrotrophic Group II C. botulinum strains are scarce; these mechanisms are of interest for food safety research and should thus be investigated. This minireview encompasses the importance of C. botulinum as a food safety hazard and its central physiological characteristics related to food-processing and storage-related stress. Special attention is given to recent findings considering genetic mechanisms C. botulinum utilizes in detecting and countering these adverse conditions.

Clostridium botulinum is a notable food pathogen and responsible for botulism due to production of botulinum neurotoxin. Strains of C. botulinum can adapt to and survive in stress conditions and food processing. The cold shock protein coding genes (csp) are involved in growth at low temperature, but they may also possess other functions. In this mutational analysis we show that cspB and cspC, but not cspA, are important for NaCl, pH and ethanol stress responses and for motility of C. botulinum ATCC 3502. In all NaCl concentrations tested, the cspB mutant had lower maximum growth rate and, together with the cspC mutant, a longer lag phase compared to the wild-type strain. At low pH, the cspB and cspC mutants showed either lower maximum growth rates or longer lag phases compared to the wild type. In all ethanol concentrations tested, the cspB mutant had lower maximum growth rates and the cspC mutant had a longer lag phase than the wild-type strain. Motility was reduced in cspA and cspC mutants, and flagella formation was affected. The results suggest that cspB plays a universal role in stress response and cspC aids C. botulinum in NaCl, pH and ethanol stress in C. botulinum ATCC 3502.

Background Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A–G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. Results Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. Conclusions This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2502-z) contains supplementary material, which is available to authorized users.

The molecular epidemiology of Listeria monocytogenes was investigated in a longitudinal study of three Finnish dairy farms during 2013 to 2016. A total of 186 bulk tank milk (BTM), 224 milk filter sock (MFS), and 1,702 barn environment samples were analyzed, and isolates of L. monocytogenes were genotyped using pulsed-field gel electrophoresis. L. monocytogenes occurred throughout the year in all sample types, and the prevalence in MFS increased significantly during the indoor season. L. monocytogenes was more prevalent in MFS (29%) than in BTM (13%) samples. However, the prevalence of L. monocytogenes varied more between farms in samples of MFS (13 to 48%) than in BTM (10 to 16%). For each farm, the L. monocytogenes genotypes detected were classified by persistence (defined as persistent if isolated from ≥3 samples during ≥6 months) and predominance (defined as predominant if >5% prevalence on at least one farm visit). The prevalence of sporadic genotypes was 4 to 5% on all three farms. In contrast, the prevalence of persistent predominant genotypes varied between farms by 4% to 16%. The highest prevalence of persistent predominant genotypes was observed on the farm with the poorest production hygiene. Persistent predominant genotypes were most prevalent on feeding surfaces, water troughs, and floors. Genotypes isolated from the milking system or from cow udders had a greater relative risk of occurring in BTM and MFS than genotypes that only occurred elsewhere in the farm, supporting the hypothesis that L. monocytogene s is transmitted to milk from contamination on the udder surface or in the milking equipment. IMPORTANCE Listeria monocytogenes is a ubiquitous environmental bacterium and the causative agent of a serious foodborne illness, listeriosis. Dairy products are common vehicles of listeriosis, and dairy cattle farms harbor L. monocytogenes genotypes associated with human listeriosis outbreaks. Indeed, dairy cattle farms act as a reservoir of L. monocytogenes , and the organism is frequently detected in bulk tank milk (BTM) and in the feces of clinically healthy cows. The ecology of L. monocytogenes in the farm environment is complex and poorly understood. Isolates of the same L. monocytogenes genotype can occur in the farm for years, but the factors contributing to the persistence of genotypes on dairy farms are unknown. Knowledge of the persistence patterns and contamination routes of L. monocytogenes on dairy farms can improve management of the contamination pressure in the farm environment and aid in the development of focused control strategies to reduce BTM contamination.

Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum , is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G+C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA , encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status.

In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II) Clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group II C. botulinum organisms are not known. Here, we show that the CLO3403/CLO3404 TCS of C. botulinum E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase ( clo3403 ) and the response regulator ( clo3404 ) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional clo3403 and clo3404 knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of clo3403 had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of C. botulinum E1 Beluga.

The two-component system CBO0366/CBO0365 was recently demonstrated to have a role in cold tolerance of group I Clostridium botulinum ATCC 3502. The mechanisms under its control, ultimately resulting in increased sensitivity to low temperature, are unknown. A transcriptomic analysis with DNA microarrays was performed to identify the differences in global gene expression patterns of the wild-type ATCC 3502 and a derivative mutant with insertionally inactivated cbo0365 at 37 and 15°C. Altogether, 150 or 141 chromosomal coding sequences (CDSs) were found to be differently expressed in the cbo0365 mutant at 37 or 15°C, respectively, and thus considered to be under the direct or indirect transcriptional control of the response regulator CBO0365. Of the differentially expressed CDSs, expression of 141 CDSs was similarly affected at both temperatures investigated, suggesting that the putative CBO0365 regulon was practically not affected by temperature. The regulon involved genes related to acetone-butanol-ethanol (ABE) fermentation, motility, arsenic resistance, and phosphate uptake and transport. Deteriorated growth at 17°C was observed for mutants with disrupted ABE fermentation pathway components ( crt , bcd , bdh , and ctfA ), arsenic detoxifying machinery components ( arsC and arsR ), or phosphate uptake mechanism components ( phoT ), suggesting roles for these mechanisms in cold tolerance of group I C. botulinum . Electrophoretic mobility shift assays showed recombinant CBO0365 to bind to the promoter regions of crt , arsR , and phoT , as well as to the promoter region of its own operon, suggesting direct DNA-binding transcriptional activation or repression as a means for CBO0365 in regulating these operons. The results provide insight to the mechanisms group I C. botulinum utilizes in coping with cold.

Clostridium botulinum is a notorious foodborne pathogen. Its ability to adapt to and grow at low temperatures is of interest for food safety. Two-component systems (TCSs) have been reported to be involved in cold-shock and growth at low temperatures. Here we show the importance of TCS CBO2306/CBO2307 in the cold-shock response of C. botulinum ATCC 3502. The relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in the late-log and stationary growth phase in relation to early logarithmic growth phase in non-shocked cultures. Importance of the CBO2306/CBO2307 in the cold stress was further demonstrated by impaired growth of insertional cbo2306 or cbo2307 knockout mutants in relation to the wild-type strain ATCC 3502. The results suggest that the TCS CBO2306/CBO2307 is important for cold-shock response and adaptation of C. botulinum ATCC 3502 to low temperature.

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.

International audience; Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.

Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39 degrees C and exposed to heat shock at 45 degrees C, followed by prolonged heat stress at 45 degrees C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.

While the quality of raw cow milk in Finland is known for its high hygienic standard, with the national average total bacterial count being below 10(4) CFU/mL annually, the prevalence of pathogenic bacteria in Finnish raw milk is underreported. The aim of this study was to determine the occurrence of Listeria monocytogenes, thermophilic Campylobacter spp., Salmonella spp., stx-positive Escherichia coli (STEC), coagulase-positive staphylococci, Yersinia spp., and Bacillus cereus group in raw cow milk samples collected from bulk tanks at 183 Finnish farms. Additionally, the hygienic quality of the milk was studied by determining the total bacterial and E. coli counts. L. monocytogenes was detected in 5.5% of the milk samples, with concentrations varying from

Bacteria have evolved a number of mechanisms for coping with stress and adapting to changing environmental conditions. Many bacteria produce small cold shock proteins (Csp) as a response to rapid temperature downshift (cold shock). During cold shock, the cell membrane fluidity and enzyme activity decrease, and the efficiency of transcription and translation is reduced due to stabilization of nucleic acid secondary structures. Moreover, protein folding is inefficient and ribosome function is hampered. Csps are thought to counteract these harmful effects by serving as nucleic acid chaperons that may prevent the formation of secondary structures in mRNA at low temperature and thus facilitate the initiation of translation. However, some Csps are non-cold inducible and they are reported to be involved in various cellular processes to promote normal growth and stress adaptation responses. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion. Therefore, Csps seem to have a wider role in stress tolerance of bacteria than previously assumed. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteropathogens that can spread through foodstuffs and cause an enteric infection called yersiniosis. Enteropathogenic Yersinia are psychrotrophs that are able to grow at temperatures close to 0oC and thus they set great challenges for the modern food industry. To be able to efficiently control psychrotrophic Yersinia during food production and storage, it is essential to understand the functions and roles of Csps in stress response of enteropathogenic Yersinia.

Outgrowth from Clostridium tyrobutyricum spores in milk can lead to butyric acid fermentation in cheeses, causing spoilage and economical loss to the dairy industry. The aim of this study was to investigate the growth of 10 C. tyrobutyricum strains at different NaCl, pH, and temperature conditions. Up to 7.5-fold differences among the maximum growth rates of different strains in the presence of 2.0% NaCl were observed. Five of 10 strains were able to grow in the presence of 3.0% NaCl, while a NaCl concentration of 3.5% was completely inhibitory to all strains. Seven of 10 strains were able to grow at pH 5.0, and up to 4- and 12.5-fold differences were observed among the maximum growth rates of different strains at pH 5.5 and 7.5, respectively. The maximum growth temperatures varied from 40.2 to 43.3°C. The temperature of 10°C inhibited the growth of all strains, while 8 of 10 strains grew at 12 and 15°C. Despite showing no growth, all strains were able to survive at 10°C. In conclusion, wide variation was observed among different C. tyrobutyricum strains in their ability to grow at different stressful conditions. Understanding the physiological diversity among the strains is important when designing food control measures and predictive models for the growth of spoilage organisms in cheese.

Summary Quantitative RT-PCR revealed that transcripts of all four putative DEAD-box RNA helicase genes of the psychrotrophic pathogen Listeria monocytogenes EGD-e are found at higher levels in organisms grown at 3°C than at 37°C. At 3°C, growth of the three corresponding gene deletion mutants Δlmo0866, Δlmo1450 and Δlmo1722 was clearly restricted. The minimum growth temperatures of the three mutants were also higher than that of the wild-type EGD-e. In addition to inability to grow at 3°C, growth of Δlmo0866 and Δlmo1722 was reduced at 25°C, suggesting special roles of Lmo0866 and Lmo1722 in growth at suboptimal temperatures. Growth of Δlmo1450 was restricted not only at 3°C and 25°C, but also at 37°C, suggesting that Lmo1450 plays a universal role in growth of L. monocytogenes EGD-e. Moreover, cold-sensitive Δlmo0866, Δlmo1450 and Δlmo1722 were impaired in motility. The Δlmo0866 and Δlmo1450 strains were non-motile, while Δlmo1722 showed reduced motility. This study shows that the putative DEAD-box RNA helicase genes lmo0866, lmo1450 and lmo1722 are necessary for cold tolerance and motility of L. monocytogenes EGD-e.

The minimum and maximum growth temperatures and the maximum growth rates at 10, 30, 37, and 40°C were determined for 24 group II Clostridium botulinum strains. Genetic diversity of the strains was revealed by amplified fragment length polymorphism (AFLP) analysis. The minimum growth temperatures ranged from 6.2 to 8.6°C, and the maximum growth temperatures ranged from 34.7 to 39.9°C. The mean maximum growth temperatures and mean maximum growth rates of type E strains at 37°C were significantly higher than those of type B and type F strains. A significant correlation between maximum growth rates at 37°C and maximum growth temperatures was found for all strains. Some type E strains with a high minimum growth temperature also had a higher maximum growth rate at 37°C than at 30°C, which suggests that some group II C. botulinum strains are more mesophilic in their growth properties than others. We found relatively small differences between AFLP clusters, indicating that diverse genetic background among the strains was not reflected in the growth properties. The growth characteristics of group II C. botulinum and some type E strains with mesophilic growth properties may have an impact on inoculation studies and predictive modeling for assessing the safety of foods.

Class I heat shock genes (HSGs) code for molecular chaperones which play a major role in the bacterial response to sudden increases of environmental temperature by assisting protein folding. Quantitative reverse transcriptase real-time PCR gene expression analysis of the food-borne pathogen Clostridium botulinum grown at 37°C showed that the class I HSGs grpE , dnaK , dnaJ , groEL , and groES and their repressor, hrcA , were expressed at constant levels in the exponential and transitional growth phases, whereas strong downregulation of all six genes was observed during stationary phase. After heat shock from 37 to 45°C, all HSGs were transiently upregulated. A mutant with insertionally inactivated hrcA expressed higher levels of class I HSGs during exponential growth than the wild type, followed by upregulation of only groES and groES after heat shock. Inactivation of hrcA or of dnaK encoding a major chaperone resulted in lower maximum growth temperatures than for the wild type and reduced growth rates under optimal conditions compared to the wild type. The dnaK mutant showed growth inhibition under all tested temperature, pH, and NaCl stress conditions. In contrast, the growth of an hrcA mutant was unaffected by mild temperature or acid stress compared to the wild-type strain, indicating that induced class I HSGs support growth under moderately nonoptimal conditions. We show that the expression of class I HSGs plays a major role for survival and growth of C. botulinum under the stressful environmental conditions that may be encountered during food processing or growth in food products, in the mammalian intestine, or in wounds.

Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe and produced by less than 5% of C. perfringens type A strains. Molecular epidemiological research in the past 15 years has focused on the reservoirs and routes of cpe-positive C. perfringens aiming to clarify the role and epidemiology of chromosomal and plasmid-borne cpe-carrying strains. This literature review highlights novel aspects in the epidemiology of CPE-mediated diseases. We suggest that (1) chromosomal and plasmid-borne cpe-carrying C. perfringens strains are genetically and epidemiologically distinct and have adapted to different environments; (2) not only chromosomal but also plasmid-borne cpe-carrying C. perfringens strains cause food poisonings; (3) other CPE-mediated diseases, such as antibiotic-associated and sporadic diarrhea, associated with plasmid-borne cpe-positive strains, may be food-related; (4) the role of animals as the main reservoir of cpe-positive C. perfringens needs to be reconsidered; (5) humans serve as an important reservoir of cpe-positive C. perfringens, introducing a contamination risk into foods through handling; and (6) the current standard procedures to diagnose C. perfringens food poisoning fail to detect and isolate many C. perfringens strains, distorting the epidemiological understanding of C. perfringens food poisoning.

The relative expression of three cold shock protein coding genes (cspA, cspB and cspC) of Clostridium botulinum ATCC 3502 was studied with quantitative RT-PCR analysis following a cold shock shift from 37 °C to 15 °C. A significant increase in the relative expression of all three genes was observed upon the temperature downshift. To validate these findings, single-gene insertional inactivation of cspA, cspB and cspC was undertaken with the ClosTron gene knock-out system. In growth experiments, mutations in cspB or cspC, but not cspA, resulted in a cold-sensitive phenotype. No growth of the cspB mutant was observed at 15°C over a ten day period, whereas at 20 °C the growth rate was 70% lower than that of wild type strain. The growth rate of cspC mutant was 70% and 80% lower than the growth rate of the wild type strain at 15 °C and 20 °C, respectively. At 37 °C the growth of cspB mutant did not differ from, but the growth rate of cspC mutant was 30% lower than, that of the wild type strain. The cspA mutant grew somewhat faster than the wild type strain at all studied temperatures. Since the inactivation of cspB resulted in the most prominent defect in growth at low temperatures, we suggest that cspB encodes the major cold shock protein of C. botulinum ATCC 3502. Understanding the mechanisms behind cold tolerance of C. botulinum helps to evaluate the safety risks this foodborne pathogen poses in the modern food industry.

The use of plastic-wrapped and nonacidified silage as cattle feed has led to an increasing number of botulism outbreaks due to Clostridium botulinum Groups I-III in dairy cattle. The involvement of Groups I and II organisms in cattle botulism has raised concern of human botulism risk associated with the consumption of dairy products. Multiplication of C. botulinum in silage and in the gastrointestinal tract of cattle with botulism has been reported, thus contamination of the farm environment and raw milk, and further transmission through the dairy chain, are possible. The standard milk pasteurization treatment does not eliminate spores, and the intrinsic factors of many dairy products allow botulinal growth and toxin production. Although rare, several large botulism outbreaks due to both commercial and home-prepared dairy products have been reported. Factors explaining these outbreaks include most importantly temperature abuse, but also unsafe formulation, inadequate fermentation, insufficient thermal processing, post-process contamination, and lack of adequate quality control for adjunct ingredients were involved. The small number of outbreaks is probably explained by a low incidence of spores in milk, the presence of competitive bacteria in pasteurized milk and other dairy products, and growth-inhibitory combinations of intrinsic and extrinsic factors in cultured and processed dairy products.

SUMMARYThe first reported bovine botulism outbreak in Finland is described. Nine out of 90 cattle on a dairy farm died after being fed non-acidified silage contaminated by animal carcasses. Type C botulinum neurotoxin gene was detected in one heifer by polymerase chain reaction (PCR) and the neurotoxin was detected by the mouse bioassay.Clostridium botulinumtype C was isolated from liver samples. The isolated strain was identified with amplified fragment length polymorphism (AFLP) analysis as group IIIC. botulinum. To our knowledge, this is the first time that a type C bovine botulism outbreak has been diagnosed by PCR and confirmed by subsequent isolation and AFLP identification of the disease strain. The importance of the acidification process in silage production to inhibitC. botulinumtoxin production in silage and thus to prevent further botulism outbreaks is emphasized. Nevertheless, preformed toxin in the carcass is not destroyed by acid.

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (

To determine the prevalence of Clostridium botulinum types A, B, E, and/or F in pig intestinal samples, different PCR-based methods were compared using artificially contaminated faeces. The methods included a multiplex PCR targeted to botulinum neurotoxin genes A, B, E, and F or a nested PCR targeted to toxin genes B, E, and F, combined with different pre-enrichment protocols and forms of templates. A method including the two-step enrichment followed by DNA extraction and multiplex PCR yielded the highest number of positives. This assay protocol was employed to investigate 100 pig intestinal samples. The sample materials studied included colon wall, intestinal content, and mucus peeled from the colon wall. Three pigs (3%) were positive for C. botulinum type B, and no other toxinotypes were detected in any sample. The number of positive samples was higher when colon wall or peeled mucus was analyzed compared to the intestinal content. C. botulinum was isolated from two PCR-positive samples and confirmed to be type B by PCR. Both isolates were shown to be proteolytic and thus to represent C. botulinum group I.

Group II (non-proteolytic) Clostridium botulinum poses a safety hazard in modern food processing, which consists of mild pasteurization treatments, anaerobic packaging, extended shelf lives and chilled storage. The high risk is reflected in the relatively large number of botulism cases due to group II C. botulinum in commercially produced foods during the past decades. Because of the high prevalence of group II C. botulinum in the environment, food raw materials may carry spores. Although group II spores are less heat-resistant than group I (proteolytic) spores, they can tolerate the heat treatments employed in the chilled food industry. Some food components may actually provide spores with protection from heat. Spore heat resistance should therefore be investigated for each food in order to determine the efficiency of industrial heat treatments. Group II strains are psychrotrophic and thus they are able to grow at refrigeration temperatures. Anaerobic packages and extended shelf lives provide C. botulinum with favourable conditions for growth and toxin formation. As the use of salt and other preservatives in these foods is limited, microbiological safety relies mainly on refrigerated storage. This sets great challenges on the production of chilled packaged foods. To ensure the safety of these foods, more than one factor should safeguard against botulinal growth and toxin production.

Humans may play a role in the transmission of gastrointestinal diseases caused by C. perfringens., We found a prevalence of 18% for enterotoxin gene–carrying (cpe+) Clostridium perfringens in the feces of healthy food handlers by PCR and isolated the organism from 11 of 23 PCR-positive persons by using hydrophobic grid membrane filter-colony hybridization. Several different cpe genotypes were recovered. The prevalence was 3.7% for plasmidial IS1151-cpe, 2.9% for plasmidial IS1470-like-cpe, 0.7% for chromosomal IS1470-cpe, and 1.5% for unknown cpe genotype. Lateral spread of cpe between C. perfringens strains was evident because strains from the same person carried IS1470-like cpe but shared no genetic relatedness according to pulsed-field gel electrophoresis analysis. Our findings suggest that healthy humans serve as a rich reservoir for cpe+ C. perfringens type A and may play a role in the etiology of gastrointestinal diseases caused by this organism. The results also indicate that humans should be considered a risk factor for spread of C. perfringens type A food poisoning and that they are a possible source of contamination for C. perfringens type A food poisoning.

The aim of this study was to investigate the occurrence of Arcobacter species in raw milk in Finland. A total of 177 raw milk samples, each from a separate farm, were examined from June to August 2011. Arcobacter species were isolated using an enrichment and selective detection procedure. Overall, 26 (15 % ) of the 177 samples yielded Arcobacter spp. Samples from 25 farms were positive for Arcobacter butzleri and from 1 farm for Arcobacter cryaerophilus. Moreover, both Arcobacter butzleri and A. cryaerophilus were recovered from 1 positive sample. To evaluate a possible genetic variability, one strain of A. butzleri from each farm and the A. cryaerophilus sample were analyzed by pulsed-field gel electrophoresis. Genotyping revealed that Arcobacter spp. populations are heterogeneous, and no dominant clone has spread in the investigated samples. Our study is the first report on the isolation of both A. butzleri and A. cryaerophilus in raw milk in Finland. Based on our findings, the presence of Arcobacter species in raw milk may pose a potential hazard for human health, in particular for consumers who prefer drinking unpasteurized milk.

The role of the alternative sigma factor SigK in cold and osmotic stress tolerance of Clostridium botulinum ATCC 3502 was demonstrated by induction of sigK after temperature downshift and exposure to hyperosmotic conditions and by impaired growth of the sigK mutants under the respective conditions.

A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying ( cpe -positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe -positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe -positive C. perfringens was successfully isolated from both of these samples. The detection of cpe -positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe- positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe- positive C. perfringens could be isolated if the ratio of cpe- positive C. perfringens spores to total C. perfringens spores was 6 × 10 −5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe -positive C. perfringens . The method also offers a new approach in the investigation of the epidemiology of cpe- positive C. perfringens strains.

Whitefish eggs were confirmed by pulsed-field gel electrophoresis to cause type E foodborne botulism in a 54-year-old patient in Finland. Botulinum neurotoxin and/or nonproteolytic Clostridium botulinum type E organisms were detected in fecal and gastric samples from the patient and in suspected whitefish eggs. Apart from C. botulinum type E, proteolytic type B organisms were detected in the patient's gastric content. This was considered to be insignificant with respect to the clinical disease, suggesting botulinal spores to be occasionally present in the human gastrointestinal tract without any apparent clinical significance. This is the first domestic case of foodborne botulism in Finland.

Clostridium botulinum produces heat-resistant endospores that may germinate and outgrow into neurotoxic cultures in foods. Sporulation is regulated by the transcription factor Spo0A and the alternative sigma factors SigF, SigE, SigG, and SigK in most spore formers studied to date. We constructed mutants of sigF , sigE , and sigG in C. botulinum ATCC 3502 and used quantitative reverse transcriptase PCR and electron microscopy to assess their expression of the sporulation pathway on transcriptional and morphological levels. In all three mutants the expression of spo0A was disrupted. The sigF and sigE mutants failed to induce sigG and sigK beyond exponential-phase levels and halted sporulation during asymmetric cell division. In the sigG mutant, peak transcription of sigE was delayed and sigK levels remained lower than that in the parent strain. The sigG mutant forespore was engulfed by the mother cell and possessed a spore coat but no peptidoglycan cortex. The findings suggest that SigF and SigE of C. botulinum ATCC 3502 are essential for early sporulation and late-stage induction of sigK , whereas SigG is essential for spore cortex formation but not for coat formation, as opposed to previous observations in B. subtilis sigG mutants. Our findings add to a growing body of evidence that regulation of sporulation in C. botulinum ATCC 3502, and among the clostridia, differs from the B. subtilis model.

Profound understanding of the mechanisms foodborne pathogenic bacteria utilize in adaptation to the environmental stress they encounter during food processing and storage is of paramount importance in design of control measures. Chill temperature is a central control measure applied in minimally processed foods; however, data on the mechanisms the foodborne pathogen Clostridium botulinum activates upon cold stress are scarce. Transcriptomic analysis on the C. botulinum ATCC 3502 strain upon temperature downshift from 37°C to 15°C was performed to identify the cold-responsive gene set of this organism. Significant up- or down-regulation of 16 and 11 genes, respectively, was observed 1 h after the cold shock. At 5 h after the temperature downshift, 199 and 210 genes were up- or down-regulated, respectively. Thus, the relatively small gene set affected initially indicated a targeted acute response to cold shock, whereas extensive metabolic remodeling appeared to take place after prolonged exposure to cold. Genes related to fatty acid biosynthesis, oxidative stress response, and iron uptake and storage were induced, in addition to mechanisms previously characterized as cold-tolerance related in bacteria. Furthermore, several uncharacterized DNA-binding transcriptional regulator-encoding genes were induced, suggesting involvement of novel regulatory mechanisms in the cold shock response of C. botulinum. The role of such regulators, CBO0477 and CBO0558A, in cold tolerance of C. botulinum ATCC 3502 was demonstrated by deteriorated growth of related mutants at 17°C.

Botulinum neurotoxin (BoNT) is produced by the anaerobic bacterium Clostridium botulinum during its vegetative growth. C. botulinum strains are commonly found in the environment, animals and in food raw materials. The bacterium forms dormant endospores, which do not produce toxins. However, the spores are highly resistant to environmental stress and thus maintain the toxic potential in environmental and agricultural systems and the food chains that occasionally provide conditions favourable for spore germination and outgrowth into a toxic culture. Besides causing botulism to man and animals, BoNTs are widely exploited by medical industries in treatment of spastic diseases. Thoroughly validated, sensitive and specific assays for neurotoxin detection are thus needed not only for laboratory diagnostics of botulism outbreaks but also for potency testing of the pharmaceutical neurotoxin products.